Pyruvate dehydrogenase (PDH) complex catalyzes the irreversible decarboxylation of pyruvic acid. The fate of acetyl-CoA produced in this reaction depends upon the metabolic context of a particular tissue. In general, well-oxygenated tissues with high demand for energy (brain, heart, skeletal muscle) use acetyl-CoA as a fuel for the Krebs cycle. In contrast, the lipogenic tissues (fat, liver, mammary gland) primarily use acetyl-CoA as a precursor for lipogenesis, i.e. to convert excess carbohydrate into triglycerides. Therefore, the regulation of PDH activity must be sophisticated enough to fulfill both catabolic and anabolic functions of the enzyme. The major working hypothesis of this proposal is that this task is accomplished through tissue-specific expression of recently discovered isozymes of pyruvate dehydrogenase phosphatase (PDH phosphatase). PDH phosphatase is a dedicated mitochondrial protein phosphatase that dephosphorylates and re-activates catalytically inactive phospho-PDH. Phosphatase is believed to mediate the effects of a variety of nutritional and hormonal stimuli on PDH activity. Thus, we suggest that catabolic and anabolic (lipogenic) tissues express different isozymes of phosphatase, which in turn, differently respond to nutritional and hormonal stimulation, by this means defining the tissue-specific responses of PDH complex. The research plan outlined in this proposal is designed to test this hypothesis. Toward this goal, we plan to study the enzymatic and regulatory properties of isozymes of PDH phosphatase. We also propose to further characterize the tissue distribution of isozymes. Finally, we will evaluate the effects of starvation and diabetes, the conditions known to be associated with down-regulation of PDH phosphatase enzymatic activity, on the expression patterns of the two isozymes. The detailed specific aims for this proposal are to: (1) determine the molecular basis for the catalysis and for the strict substrate specificity of pyruvate dehydrogenase phosphatase; (2) further characterize the molecular factors responsible for the regulation of catalytic activity of the isozymes of phosphatase; (3) evaluate the contribution of each isozyme to the tissue specific regulation of the activity of pyruvate dehydrogenase complex; and (4) elucidate the molecular mechanisms responsible for the stable changes in phosphatase activity observed in starvation and diabetes. The accomplishment of these objectives will shed new light on the role of PDH reaction in the regulation of general carbohydrate and lipid metabolism. In the long run, it may lead to the development of highly specific drugs that will alleviate complications associated with diseases like diabetes, ischemia and sepsis.